I am struggling to interpret an indirect ELISA I am making which I would appreciate your thoughts please! It involved coating wells with plasma overnight, blocked with milk the next day, followed by primary ab, then detection ab and finally the substrate (I used ALP-PNPP system).

I got nice reproducible standard curves and uniformly negative signals from BSA. So all good.

However I am seeing a dose-dependent increase in signal in plasma samples. As plasma dilution increased, signal increased - in other words, the more dilute the plasma (so in theory lesser amount of antigen) - the stronger the signal! I could not understand why. I repeated the experiments and I am seeing it every time (4x now).

At first I thought it could be due to primary ab binding to "unoccupied sites in the wells" directly, which then generated a signal when secondary added, which would explain why the signal is stronger in diluted sample (as more sites for primary ab to bind non-specifically). But if that's the case I should see that in the BSA wells, but I am not. I did block the wells with milk after coating to ensure all unbound sites are occupied.

Then I thought could it be something in the plasma which is inhibiting the substrate reaction (ALP)? So the more dilute the plasma, the less the inhibition, and the stronger the signal. But plasma was only used to coat the plate overnight. The plates were then washed after every step with TBS-tween (2-4 times) after every step. So I would imagine not much of any “inhibitors” are around by the time substrate is added.

I also tried mixing plasma directly with the antibody in a separate experiment, and signal was not diminished, thus argued against an inhibition.

Then I thought whether it could be a “hook” effect. In other words, the plasma is too concentrated and antigens are crowding up thus reducing the amount of binding. But I thought usually plasma then need to be diluted up to 100 folds to relieve the hook. But I am seeing a “nice” dose dependent increase in signal as plasma was diluted from 1:2, 1:4, 1:8 to 1:16 only.

I don't understand the format of your ELISA. Typically, coating is performed using a purified preparation of antigen in a non-complex buffer (PBS or Carbonate for example). Although I have heard of direct coating using complex buffers (including plasma) for very small peptides, this is uncommon.

If your coating solution contains plasma, it is very unlikely that you will end up with any specific coating because the plasma components will effectively block the plate surface from further binding.

Your setup is new to me.
Could you specify what you intend to measure?
As Elisadeveloper mentions coating with purified antigen is appreciated, and if you want to measure some plasma protein, incubated dilutions of your sample (e.g. plasma) with fixed concentrations of your antibody. If antigen levels in your plasma is high, low results will be obtained, because of competitive binding of the antibody to the sample antigen.
Hope this helps
PeterJallerup